P724: THE BONE MARROW MESENCHYMAL CELL-DERIVED EXTRACELLULAR MATRIX IS SIGNIFICANTLY ALTERED IN PATIENTS WITH MELODYSPLASTIC SYNDROMES
Authors: A. Bains, L. Behrens Wu, M. Richter, V. Magno, S. Rother, M. Cross, C. Werner, M. Wobus, U. Platzbecker
Journal: HemaSphere (2022)

Background: Myelodysplastic syndromes (MDS) are characterised by ineffective hematopoiesis and peripheral cytopenia in one or more hematopoietic lineages. MDS is driven by genetic and epigenetic lesions in the hematopoietic clone itself, but also by changes in the bone marrow microenvironment. Here, the mesenchymal stromal cells (MSCs) regulate hematopoietic cell behaviour through cell-cell contact, paracrine signalling and metabolic support. The MSC-derived Extracellular matrix (ECM) plays a key role in coordinating both physical and biochemical interactions but the involvement of ECM in MDS has yet to be determined. Aims: We aimed to characterise the relevant parameters of the mesenchymal cell-derived ECM generated by MSCs from healthy donor and MDS patient bonemarrow. Methods: MSC from low risk (LR) and high risk (HR) MDS patients and age-matched healthy donors were cultured for 10 days in DMEM Glutamax medium supplemented with 10% FBS on glass slides pre-coated sequentially with poly-octadecene-alt-maleic anhydride (POMA) and human fibronectin. The confluent monolayers were washed in PBS, decellularized by washing with 20mM ammonia and were subject to DNase treatment. The resulting ECM were stored for up to 4 weeks at 4oC. Matrix structure was analysed by scanning electron microscopy (SEM) and atomic force microscopy (AFM), glycosaminoglycan (GAG) content by staining with peanut and wheat germ agglutinin and collagen composition by immunostaining with antibodies to collagens 1 and 4. Total collagen content was quantified by Sircol assay. In parallel, the expression of matrix-related genes was measured by real time PCR of RNA extracted from the MSCs. Results: In SEM, the ECM network generated by MSCs derived from both low risk (LR) and high risk (HR) MDS was thicker and of a higher density than that produced by MSCs from healthy donors. AFM analysis showed ECM of MDS-derived MSCs also had a significantly reduced rigidity. An increase in overall collagen content in MDS-derived ECM was detected by sircol assay and confirmed by immunostaining for the most prevalent collagen subtypes 1 and 4. Lectin staining revealed disease stage-specific differences in GAG composition: The levels of both GAGs carrying N-acetyl glucosamine and those carrying N-acetyl galactosamine sugars were increased in ECM from LR-MDS, while ECM from HR-MDS retained high levels of N-acetyl glucosamine but carried only low levels of N-acetyl galactosamine GAGs. Finally, the pattern of hyaluronan synthase (HAS) gene expression differed markedly between MSCs from healthy donor, LR- and HR-MDS. Specifically, HAS1 was strongly increased in LR-MDS and remained significantly above control levels in HR-MDS, while HAS3 was down-regulated specifically in HR-MDS. Consistent with this, increased levels of low molecular weight hyaluronic acid was observed in ECM from LR-MDS Summary/Conclusion: We report both structural and compositional differences between the MSC-derived ECM from healthy donors, LR-MDS and HR-MDS. The thicker and softer structure of the MDS matrix may be both a consequence and a driver of changes in the MSC phenotype. Increase in the major collagens 1 and 4 are consistent with an increase in overall ECM, but it remains unclear whether the decreased rigidity of ECM in MDS is due to differences in protein cross-linking or in the levels of hydrated glycosaminoglycans. MDS-associated changes in hyaluronic acid expression suggest that MSC-derived HA may contribute to the inflammatory bone marrow state characteristics of LR-MDS.